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Originally published as Biophys J. BioFAST on August 4, 2006.
doi:10.1529/biophysj.106.090993
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Biophysical Journal 91:3829-3840 (2006)
© 2006 The Biophysical Society

Impact of the Mutation A21G (Flemish Variant) on Alzheimer's ß-Amyloid Dimers by Molecular Dynamics Simulations

Alexis Huet and Philippe Derreumaux

Laboratoire de Biochimie Théorique, UPR 9080, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, et Université Paris, Paris, France

Correspondence: Address reprint requests to P. Derreumaux, Tel.: 33-1-58-41-50-16; E-mail: philippe.derreumaux{at}ibpc.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Soluble oligomers of the amyloid ß-protein (Aß) are linked to Alzheimer's disease. Irrespective of the nature of the nucleus before fibril growth, dimers are essential species in Aß assembly, but their transient character has precluded, thus far, high-resolution structure determination. We have investigated the effects of the point mutation A21G on Aß dimers by performing high temperature all-atom molecular dynamics simulations of Aß40, Aß42, and their Flemish variants (A21G) starting from their fibrillar conformations. Aß dimers are found in equilibrium between various topologies, and the absence of common structural features shared by the four species makes problematic the design of a unique inhibitor for blocking dimers. We also show that the impact of the point mutation A21G on Aß structure and dynamics varies from Aß40 to Aß42. Finally, we provide a possible structural explanation for the reduced aggregation rate of Aß fibrils containing the Flemish disease-causing mutation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The formation of amyloid fibrils is a hallmark of many human diseases and results from the misfolding of proteins into cross ß-sheet structure (1Go). Alzheimer's disease, for instance, is characterized by deposition of amyloid fibrils in the brain parenchyma and cortical blood vessels. This accumulation consists of 40- and 42-mer peptides (Aß40 and Aß42) produced through endoproteolysis of the ß-amyloid precursor transmembrane protein by ß- and {gamma}-protease (2Go). Thus far, a high-resolution structure for Aß40 and Aß42 is not available, in contrast to a seven-residue peptide fragment from yeast protein Sup35 (3Go), but we know that the ß-strands run perpendicular to the fiber axis, the hydrogen-bonding interactions run parallel to the fiber axis, and the chains are in parallel register. Several models have been proposed, based on solid-state NMR experiments (4Go,5Go), hydrogen/deuterium (H/D) exchanges measurements with (6Go) or without mutagenesis data (7Go), and proline scanning methods (8Go,9Go). All models share a disordered N-terminal region spanning at least residues 1–10, and differ in the number and length of strands and loops, and in the network of intermolecular hydrogen-bonding and sidechain-sidechain interactions.

The kinetic model, by which the Aß peptides aggregate into amyloid fibrils, is believed to follow a nucleation-growth model, with a lag-phase of several days. Oligomerization is very sensitive to amino-acid variations. Residues 41 and 42 affect the characteristics of the nucleus (10Go); Aß42 forms fibrils at a higher rate than Aß40 and the Alzheimer's disease-causing A21G (Flemish) mutation (11Go) has a slower aggregation kinetic than wild-type Aß and the E22Q (Dutch) (12Go,13Go), E22K (Italian) (14Go), E22G (Arctic) (15Go), and D23N (Iowa) (16Go) mutations (17Go,18Go).

In contrast to the late aggregates or protofibrils which are well characterized, the structures of the oligomers forming in the early steps of aggregation are poorly understood because they are transient in character. Although dimers are in equilibrium with higher-order species before the formation of the nucleus, dimer formation is certainly critical in Aß assembly. However, much remains to be elucidated regarding the structure of these dimers, which are sufficient for toxicity (19Go,20Go), from both the experimental and theoretical fronts. We know, however, that Aß40 and Aß42 form stable dimers in solution using fluorescence resonance energy transfer (21Go–23Go). The stability of several Aß species in dimers and higher-order mers has been investigated by explicit solvent molecular dynamics (MD) simulations, including dimers of Aß10–35 (24Go), hexamers of Aß15–36 (25Go,26Go), and octamers of Aß9–40 (27Go). These studies at 300 K, however, explore local fluctuations around preformed arrangements. The assembly process of Aß1–40 and Aß1–42 dimers was studied by discontinuous MD (DMD) simulations, and planar ß-strand Aß dimers were found instable (28Go). Similarly, the aggregation process of multimers of Aß40 and Aß42 (29Go) has been explored using DMD simulations, but the results remain to be confirmed using a more elaborated chain representation and force field (29Go).

In this study, we have investigated the effects of the point mutation A21G on Aß dimers by performing all-atom molecular dynamics simulations of Aß9–40, Aß9–42 and their Flemish variants (A21G) at 400 K starting from their fibrillar conformations. Note that omission of residues 1–8 does not prevent the peptide from forming fibrils (30Go). The point mutation A21G is interesting because no mechanistic explanation has been offered for its effect on aggregation rate, in contrast to the mutations at positions 22 and 23 (31Go,32Go). We emphasize that our goal is not to determine the equilibrium structures of Aß dimers. This is currently out of reach by using high temperature MD simulations or replica exchange simulations with current computer facilities. Rather, our aim is to understand at an atomic level of detail the effects of the mutation A21G on fibrillarlike dimeric structures of Aß40 and Aß42.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Differences in Aß40 and Aß42 fibril models
The Aß42 sequence is

Formula
with the amino acids 21 underlined and 41 and 42 in bold. Several fibril models have been proposed. Both Aß40 models of 2002 (4Go) and 2006 (5Go) models of Petkova et al. are based on solid-state NMR measurements, but the 2006 model includes measurements that provide direct constraints on quaternary structure. The 2005 Aß42 model of Luhrs et al. (6Go) is based on the solid-state NMR measurements carried by Petkova et al. in 2002, and on their own H/D exchange and mutagenesis data. Olofsson et al. (7Go) have also performed H/D exchange measurements on Aß42 fibrils. All these models show a cross-ß structure with parallel ß-sheets, but significant differences are identified.

  1. Residues 1–8 are disordered in the 2002 and 2006 Aß40 models, versus residues 1–15 in the 2005 Aß42 model proposed by Luhrs et al. (6Go). As the result, ß-strand S1 encompasses residues 12–24 in the Aß40 models versus 18–26 in the Aß42 Luhr's model, and ß-strand S2 covers residues 30–40 and 31-42, respectively. In contrast, Olofsson et al. (7Go) found that residues 11–24 are highly protected in Aß42, in agreement with the Petkova's Aß40 models, so the two fibril structures may actually be very similar.
  2. The salt bridge between residues Asp23 and Lys28 and the side-chain interactions are intramolecular in the 2002 Aß40 model, but intermolecular in the 2005 Aß42 model. The inter-ß-sheet side-chain interactions are between the odd-numbered residues of S1 and S2 in the 2002 Aß40 model, and between the odd-numbered residues of S1 and the even-numbered residues of S2 in the 2005 Aß42 model. However, the revisited 2006 Aß40 model indicates intermolecular Asp23–Lys28 salt bridges and contacts between odd residues in S1 with even residues in S2.

These analyses, with the proposed model of Williams et al. (8Go), indicate that the fibril structures are likely to evolve as additional experimental data become available, but such a refinement is very complicated since the dimensions and morphologies of fibrils vary with solution conditions and degrees of agitation (33Go).

Dynamics simulations
MD simulations are performed at neutral pH using the program GROMACS2.0 and the all-hydrogen energy function GROMOS96 (34Go). The starting point is the 2002 NMR solid-state structure of Aß40 fibril (4Go), which lacks the atomic positions of residues 1–8. Note that both the Aß42 model and the 2006 Aß40 model were unknown at the beginning of this work. The initial structures for Aß42 and the A21G variants are constructed using the SWISS-MODEL server (35Go).

All Aß models are solvated in a rectangular box of 90, 50, and 40 Å sides with ~6000 simple point-charge water molecules and simulated using periodic boundary conditions. The particle-mesh Ewald method is used with a cutoff distance of 12 Å. Aß models are minimized by 300 steps of steepest descent and 600 steps of conjugate gradient and then equilibrated at the desired temperature for 80 ps under C{alpha} atom restraints followed by 80 ps free of any atomic restraints. Subsequently, MD simulations are performed in the canonical NPT ensemble. The time step for dynamics is 2.0 fs using the LINCS algorithm and the list of nonbonded interactions is updated every 20 fs. Temperature is controlled using a weak coupling to a bath of constant T (coupling time of 0.1 ps) and pressure by a weak coupling to a bath of constant P (1 atm, coupling time of 0.5 ps).

All Aß models are simulated for 10 ns at 400 K to increase phase space sampling. This is an advisable choice in contrast to the standard temperature (500 K) to induce unfolding and conformational changes. The Van Gunsteren group (36Go) showed that the use of temperatures higher than 400 K is very likely to affect the unfolding, kinetics, and thermodynamics of proteins. Dinner and Karplus (37Go) studied thermal folding and unfolding of lattice proteins and found that unfolding is the reversal of fast-track folding. Klimov and Thirumalai (38Go) moved one step further and showed that folding pathways at 300 K and unfolding pathways at 390–420 K are similar using off-lattice models. It follows that the present paths at 400 K are closely related to the conformational changes at 300 K, although the population of each dominant unfolded state changes with T. The Aß40 and its A21G variant are also simulated starting from the same structure using different initial velocities. All runs took 80 days on a cluster of five 1.5-GHz processors.

The trajectories are analyzed using several order parameters. These include the C{alpha} root mean-square (RMS) deviations from the minimized NMR structure and the C{alpha} RMS fluctuations (RMSF) relative to the average MD structure. We also follow the evolution of the radius of gyration Rg, the end-to-end distance, and the percentage of secondary structure content using the STRIDE program (39Go). Because the Aß models differ in length, we only use residues 9–40 to compare the trajectories. The MD-generated structures are also clustered using a C{alpha} RMS deviation cutoff of 3 Å, and analyzed by their contact maps and percentages of native contacts. Here, native refers to the 2002 Aß40 solid-state NMR conformation, and a contact is defined when aliphatic carbon atoms of two nonsequential side chains come within 5.4 Å or any other atom of two nonsequential side chains lies within 4.6 Å (40Go). The structural models are drawn by using the VMD software (41Go).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Conformational stability of dimers
The distributions of the C{alpha} RMS deviations (RMSDs) of residues 9–40 from the minimized structures of monomers A and B are displayed in Fig. 1, a and b, for all Aß models. Aß40 is much less flexible than Aß42 and the mutants. The C{alpha} RMSD population of monomer A is essentially centered at 0.5 nm in Aß40, and varies between 0.5 and 0.9 nm in Aß42-A21G, 0.7 and 1.1 nm in Aß42, and between 0.7 and 1.25 nm in Aß40-A21G. The variability in conformations can be further explored by the distributions of the end-to-end C{alpha} distances between residues 9 and 40 in Fig. 1, c and d. In Aß40, the most populated end-to-end distance is centered at 2 nm in monomer A and spans larger distances in monomer B, which is in contrast with Aß42, where the end-to-end distance fluctuates between 0.8 and 2.8 nm in both monomers. Higher stability of Aß40 is also seen in the evolution of the radius of gyration (Rg) with time in Fig. 1 e and the distributions of Rg in Fig. 1 f. In Aß40, the MD-averaged Rg is 1.5 nm to be compared to the NMR value of 1.75 nm, and the Rg distribution has a well-defined peak. This contrasts with Aß42 and the mutants which have a lower mean radius of gyration (1.1 nm in Aß40-A21G, Fig. 1 e) and larger standard deviations of Rg (Fig. 1 f).


Figure 1
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  		FIGURE 1  Dynamical properties as a function of Aß species. Distributions of the RMSDs from the minimized structures: (A) monomer A and (B) monomer B. Distributions of the C{alpha}–C{alpha} distances between residues 9 and 40: (C) monomer A and (D) monomer B. Radius of gyration as a function of time (E) and distributions of the radius of gyration (F); both panels use the dimer of each species. RMSFs from their mean structures: (G) monomer A and (H) monomer B. Distributions of the solvent-exposed surface area: (I) main-chain atoms of residues 9–40 and (J) side-chain atoms of residues 9–40; both panels use the dimer of each species. Four colors are used to distinguish the species: green (Aß40); blue (Aß40-A21G); black (Aß42); and red (Aß42-A21G).

 
The RMSFs, with respect to the average MD structure in Fig. 1, g and h, show high flexibility at the N-terminal and C-terminal extremities for all models, in agreement with MD studies of a dimer of Aß10–35 (24Go) and eight Aß40 peptides (27Go). The largest differences in RMSF between all Aß species are found in the loop between S1 and S2 (residues 21–30, RMSF of 0.3 Å in Aß40 versus 0.6 Å in other species, Fig. 1 g), and in strand S2 (notably for monomer B, Fig. 1 h). These regions match the locations where experimental data show the lowest structural determination.

The structural fluctuations, using the RMSD from the minimized and MD-averaged structures, the end-to-end distance, and the radius of gyration show that monomer A is less flexible than monomer B. This asymmetry in structural fluctuations of the monomers in a homodimer has been discussed in 10-ns MD simulations on decoys of Aß10–35 at 300 K (24Go). Asymmetric conformational changes were also observed by lattice (42Go) and off-lattice (43Go) aggregation MC simulations of homodimer amyloid-forming models. The effect of this asymmetry on fibril nucleus formation, remains, however, to be determined.

Overall, we see that the flexibility increases in the following order: Aß40 -> Aß42 A21G -> Aß42 and Aß40-A21G. This conclusion is also supported by the analysis of the solvent-exposed surface area, using the method of Lee and Richards (44Go), although the role of water molecules on dimer unfolding changes according to different sequences. The backbone atoms of residues 9–40 are more exposed to the solvent in Aß42 and Aß40-A21G than in Aß40 (Fig. 1 i). The side-chain atoms of residues 9–40 are more exposed in Aß40 than in Aß42 and the Aß variants (Fig. 1 j). (Identical results are obtained excluding residue 21.) This change in solvent accessibility does not have a one-to-one mapping with the amino-acid number. For instance, the distributions of solvent accessibility of the Asp22 side chain match exactly in all simulations, whereas those of Asp11 side-chain change (data not shown).

To investigate whether these results vary with different initial velocity distributions, the simulations of Aß40 and Aß40-A21G are repeated for 10 ns using the same starting structure. The RMSD between all pairs of structures is determined using a pool of 1000 structures taken at 10-ps intervals. By calculating for each Aß species the number of pairs of structures as a function of the RMSD deviation, we see that the RMSD plots superpose well from one run to another for Aß40 (Fig. 2 a) and Aß40-A21G (Fig. 2 b). Consistency between the runs is also seen in the C{alpha} RMSD from the minimized structure in Fig. 2 c and the evolution of the radius of gyration with time in Fig. 2 d. Fig. 2, e and f, show the RMSF profiles of monomers A and B in Aß40-A21G. The RMSFs are very similar in monomer A, but differ by 2 Å in the 25–32 region of monomer B. Overall, these analyses indicate that different simulations on the same sequence produce equivalent results.


Figure 2
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  		FIGURE 2  Effects of two different initial velocities on Aß40 and Aß40-A21G properties. Number of pairs of structures as a function of the RMSD for Aß40 runs (A) and Aß40-A21G runs (B). RMSD (C) and radius of gyration (D) as a function of time for the dimers of Aß40-A21G. RMSFs from their mean structures for the monomer A (E) and the monomer B (F) of Aß40-A21G. Solid lines are used for the first seed and dotted lines for the second seed.

 
Effects on secondary structures and loop conformations
Table 1 gives the mean percentage and standard deviation of {alpha}-helix, ß-strand, and turn and random coil contents, using residues 9–40 for all species. As expected from high temperature simulations ignoring interactions with neighboring protofilaments, the percentage of ß-strand decreases, but the variation with Aß length and mutation A21G is rather surprising. Note that the percentage of ß-strand is 83% (26:31) in the NMR-model of Aß9–40. The MD-averaged percentage of ß-strand is 46% in both Aß40 and Aß42-A21G, and 37% in Aß40-A21G and Aß42. The variation in ß-strand content does not result from an increase in {alpha}-helix (its population amounts to a few percent) and turn content, but in random coil conformations.


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  		TABLE 1  Secondary structure compositions of Aß40, Aß42 and their A21G variants from explicit solvent MD simulations at 400 K

 
To quantify the effects of extending or mutating Aß40 on secondary structures, we calculate the probability of each residue to be assigned with ß-strand character along the trajectories. We see that, in Aß40, the strands S1 and S2 are rather well-conserved spanning residues 11–20 and 30–36 in both monomers (Fig. 3 a), whereas in Aß42 and the mutants, monomer B is clearly less ß-prone than monomer A, and the details vary from one Aß species to another. For instance, the reduction of ß-content in Aß40-A21G results from a shorter strand S1 in monomer B spanning residues 12–18 (but not monomer A, Fig. 3 b), and shorter strands S2 in monomers A and B. In contrast, the reduction of ß content in Aß42 comes from a shorter strand S2 in monomer A and broken strands S2 in monomer B (see the minimum at positions 37–38 in Fig. 3 c). Finally, the combined impact of A21G and residues 41–42 on ß-strand percentage is rather complex: they shorten strand S1 (monomer B) or extend it by four residues (monomer A); they break strand S2 with turn preferences at positions 37–38 (monomer B) or extend it at positions 39–41 (Fig. 3 d).


Figure 3
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  		FIGURE 3  MD-averaged probability of ß-strand occurrence along the amino-acid sequence. Aß40 (A), Aß40-A21G (B), Aß42 (C), and Aß42-A21G (D). The location of the ß-strands S1 and S2 within the 2002 Aß9–40 model and the 2005 Aß17–42 model are indicated by thick and thin solid lines, respectively. In each panel, solid lines are used for the monomer A and dotted lines for the monomer B.

 
To determine why the mutation A21G destabilizes ß-sheets in Aß40 but not in Aß42, in Fig. 4 we plot the Ramachandran plot of the residue 21 from all MD-generated structures of each Aß model. We see that in Aß40 and Aß42, Ala21 explores essentially the favored ({phi},{psi}) regions with a high density of residues with ß-character (Fig. 4, a and c). On the other hand, in Aß40-A21G, the Ramachandran plot of Gly21 has a typical pattern of a glycine (Fig. 4 b), whereas in Aß42-A21G, the Ramachandran plot resembles that of a standard residue (Fig. 4 d). These results indicate that the residues 41 and 42 have a strong impact on the flexibility of the residue Gly at position 21, and may affect the loop-spanning residues 21–30.


Figure 4
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  		FIGURE 4  Ramachandran plot of residue 21. (A) Aß40, (B) Aß40-A21G, (C) Aß42, and (D) Aß42-A21G. Dihedral angles {phi} and {psi} are reported using both monomers.

 
To quantify the variations of this loop, we calculate the intramolecular and intermolecular distances between the N{zeta} atom of Lys28 and C{gamma} atom of Glu22 and Asp23. Salt bridges are considered formed if they come within 4.2 Å (45Go). The formation times of all salt-bridges during the trajectories are reported in Table 2. As expected, we find that the native intramolecular Asp23-Lys28 salt bridge is populated in all models, although to a lesser extent in Aß42 (34% of the time) compared to Aß40 (52% of the time). Note that A21G reduces the occurrence of this salt bridge: 32% of the time in Aß40-A21G and 22% of the time in Aß42-A21G. Similarly, the nonnative intramolecular Glu22-Lys28 salt bridge is also populated in all models: 10.7%, 9.7%, 14.8%, and 15.8% for Aß40, Aß42, Aß40-A21G, and Aß42-A21G, respectively. The finding of two distinct Coulombic interactions involving Lys28 are fully consistent with recent NMR experiments of the monomer of the fragment Aß21–30 (31Go). Interestingly, the intermolecular salt bridge between Asp23 of monomer B and Lys28 of monomer A, one fingerprint of the 2005 Aß42 model and the 2006 Aß40 model, is also populated in all simulations, and notably in Aß40 (22.6% of the time) and Aß42-A21G (13.4% of the time).


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  		TABLE 2  Formation time percentage of intramolecular and intermolecular salt bridges between the residues Glu22 and Lys28 and between the residues Asp23 and Lys28

 
Geometrical characteristics of final Aß dimers
The centers of the most populated clusters within 7.5–10 ns are shown in Fig. 5. The populations of these clusters vary from 40% (Aß40-A21G) to 80% (Aß42-A21G). The sidechain-sidechain contact map of each final topology is given in Fig. 6.


Figure 5
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  		FIGURE 5  Final structures and topologies of Aß dimers. (A) The initial dimer of Aß40 used for the simulations, and the location of the residues His14, Phe19, Glu22, Asp23, Lys28, and Met35. The center of the most populated clusters within 7.5–10 ns are shown for Aß40 (B), Aß40-A21G (C), Aß42 (D), and Aß42-A21G (E). The TOPS cartoon of each topology is also given and full explanation of the cartoons can be found at http://www3.ebi.ac.uk/tops/ExplainSummary.html. In brief, monomer A runs from NA to CA, and monomer B from NB and CB. Triangular and circular symbols represent the ß-strands and {alpha}-helices, respectively. Triangles on the same horizontal row indicate hydrogen-bonding interactions between the strands. In all panels, residues 9–25 are in blue within monomer A and in red within monomer B; residues 26–30 are in white, and residues 31–40 (or 31–42) are in gray within monomer A and in orange within monomer B.

 

Figure 6
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  		FIGURE 6  Sidechain-sidechain contact maps of Aß dimers. (A) Initial Aß40, (B) final Aß40, (C) final Aß40-A21G, (D) final Aß42, and (E) final Aß42-A21G maps for the structures shown in Fig. 5. Each Aß species is described by four panels: panels AA and BB show the intramolecular contacts within monomers A and B, respectively; panels AB and BA show the intermolecular interactions between monomers A and B, and between B and A, respectively. A diagonal dotted line is drawn to separate the native (upper) from nonnative (lower) interactions, and a vertical and horizontal dotted line indicates the position of the residue Met35. The criteria used for defining a contact are described in Materials and Methods.

 
As a result of unfolding, the percentage of native sidechain-sidechain contacts averages 40% in Aß40, 22% in Aß42, 20% in Aß42-A21G, and 19% in Aß40-A21G, and all final structures adopt nonnative topologies, as described by TOPS cartoons (46Go). In what follows, we analyze, for each Aß species, the topological reorganization, the salt bridge within the (21Go–30Go) loop, and the intra- and intermolecular interactions involving notably Met35, Phe19, Phe20, Ile41, and Ala42 (5Go). We recall that, in the starting Aß40 structure shown in Fig. 5 a, residues i of monomer A are in contact with residues i of monomer B, both intramolecular and intermolecular contacts are formed between Phe19 and Met35, and between His13 and Ile41 (Fig. 6 a, panel AB).

The final topology of Aß40 in Fig. 5 b is a planar four-stranded ß-sheet with native parallel registers between the strands S1 and between the strands S2, and nonnative antiparallel hydrogen-bonds between S2 of monomer B and S1 of monomer A. S1 encompasses residues 11–20 in both monomers, S2 spans residues 31–38 in monomer A and residues 31–35 in monomer B. This structure, which lacks the salt bridge between Asp23 and Lys28, has replaced the native intramolecular contacts between the strands S1 and S2 (Fig. 6 b, panels AA and BB) by nonnative intermolecular contacts between residues 34–40 of monomer A and residues 14–20 of monomer B (Fig. 6 b, panel BA).

Aß40-A21G in Fig. 5 c is also a four-stranded ß-sheet with the strands S1 in parallel register, but the strands S2 are separated by 15 Å from each other and folded back against their strands S1. Strand S1 spans residues 13–19 and 13–16 in monomers A and B, whereas strand S2 covers residues 35–38 in monomer A and 34–35 in monomer B. The interface between the chains is essentially nonnative (Fig. 6 c, panels AB and BA), although the intramolecular intermolecular Phe19 and Met35 is preserved, and is characterized by a cluster of interactions between Met35, and eight residues at positions 17, 19, 20, 23, 27, 28, and 31 (Fig. 6 c, panel AA).

The final topology of Aß changes drastically with the presence of residues Ile41 and Ala42. Aß42 is essentially characterized by a seven-stranded ß-sheet, although a short {alpha}-helix occurs at positions 22–26 in monomer A (Fig. 5 d). The strands cover residues 14–20, 26–27, 30–31, and 36–41 in monomer A and residues 10–20, 32–33, and 38–40 in monomer B. Two salt bridges are formed: one between Glu22 and Lys16 within monomer B and the other between Asp23 of monomer B and Lys28 of monomer A. Met35 of monomer A is in contact with Phe19 of both monomers. In contrast to Aß42, Aß42-A21G leads to a five-stranded ß-sheet with mixed parallel and antiparallel strands (Fig. 5 e). Strands encompass residues 11–27 and 29–39 in monomer A, and residues 13–18, 25–33, and 39–42 in monomer B. The intramolecular salt bridge between Glu22 and Lys28 is only formed in monomer B, and Met35 is in contact with Phe19 and Phe20 of monomer A.

It is of interest to determine the native interactions and the structural features that are shared by all four Aß species. We find that only the native intermolecular contact between His14 residues is conserved in all species. All final Aß dimers disrupt the interfaces between the strands S1 and S2, and between the strands of S2, but tend to preserve the interactions between the central hydrophobic clusters (CHC) spanning residues Leu17-Ala21, although the contacts observed are not conserved. For instance, the native intermolecular interaction between Phe19 residues is conserved in Aß40, Aß42, and its variant, but not in Aß40-A21G.

Comparison with in vitro and previous in silico experiments
Solution NMR studies of Aß10–35 (47Go), Aß1–40, and Aß1–42 (48Go) showed that CHC is the most structured region. Similarly, MD simulations of Aß10–35 in monomeric (49Go) and dimeric (24Go) forms showed that the CHC region is much less flexible than the rest of the protein. We find that this region, which is known to be essential for aggregation (50Go), is well conserved in wild-type Aß40 and Aß42, but not in their variants, strands S1 covering residues 13–16 and 13–18 in the monomers B of Aß40-A21G and Aß42-A21G, respectively.

The residues Ile41 and Ala42 are known to play a crucial role in Aß oligomerization. Moritomo et al. (9Go) found that the Aß42 I41T and A42T mutants aggregate potently, and proposed that the hydrophobicity of the C-terminal two residues of Aß42 is not related to its aggregative ability, and that the C-terminal three residues adopt the ß-sheet.

Hou et al. (48Go) measured 1H{alpha}, 13C{alpha}, and 13Cß chemical shift indices of the Aß1–40 and Aß1–42 monomeric species and found that the C-terminus of Aß1–42 has a propensity for ß-sheet structure, whereas that of Aß1–40 has not. Lazo et al. (31Go) coupled limited proteolysis and mass spectrometry and found that the residues Val39-Ala42 are protease-resistant in Aß1–42, while the residues Val39-Ala40 are not in Aß1–40. We find that the residues 41 and 42 have two major effects on the structure. Firstly, they favor the extension and formation of ß-strand at position 39–42. Secondly, they disrupt the native interface between the chains and enhance the number of intramolecular interactions, irrespective of the amino acid at position 21. As seen in panels AA and BB of Fig. 6, d and e, both Aß42 and Aß42-A21G species display two new domains (encircled) that are absent in the Aß40 species: one involving residues 13–27 and 29–42 in monomer A, the other involving residues 10–17 and 37–42 in monomer B. In particular, Aß42 and its mutant display de novo intramolecular interactions between (Ile41, Ala42) and (His14, Gln15, Lys16, Val18, and Phe19). In addition, we see that Met35 contacts Val40 in Aß42 and its A21G mutant, whereas Met35 does not contact the C-terminal residues in Aß40 and its A21G mutant. This difference in Met35 contacts between the Aß40 and Aß42 species is made possible by the formation of a turn centered at Gly37-Gly38 in Aß42, but not in Aß40 (see Fig. 3). Such a finding is fully consistent with DMD simulations of multimers (29Go) and MD simulations of a monomer (51Go).

Several studies have emphasized the importance of the intramolecular salt bridge between residues 23 and 28 in fibril formation (5Go,33Go). An engineered lactam bridge between Asp23 and Lys28 increases the Aß1–40 fibrillogenesis rate by three orders of magnitude (52Go). Solution NMR study and DMD simulations of the fragment Aß21–30 also suggested a possible mechanism for the effects of mutations at positions 22 and 23, based on a change of the populations of the salt bridges 23:28 and 22:28 (31Go,32Go). In contrast, the NMR fibril model of Aß17–42 points to an intermolecular salt bridge between residues 23 and 28. Our simulations provide strong evidence of the existence of all these salt bridges in equilibrium, whose populations are determined by the presence of Ile41 and Ala42, and the nature of the amino acid at position 21.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
We have studied the impact of the point mutation A21G on the structure of Aß dimers, by using a total of six unfolding molecular dynamics simulations at 400 K. All simulations of Aß40, Aß42, and their variants start from the conformation within the NMR 2002 fibril model of Aß40. Although this structure differs from the 2005 and 2006 models, it is a reasonable starting point because there is strong evidence that a unique structure does not exist, polymorphism likely reflecting multiples alternatives for the packing and interactions within the core of the fibril (33Go). In addition, we are only interested in the qualitative effect of Flemish mutation on the stability of different Aß dimers and on the kinetics of assembly. Quantitative analysis is beyond the scope of this work for two reasons. The first reason is that a direct comparison between conformational distributions from experiment and simulation is difficult. Circular dichroism and NMR studies of low molecular weight Aß, consisting of monomers and dimers in equilibrium with multimers as large as heptamers, give 60–80% of random coils, 10–20% of ß-strand, and <10% of {alpha}-helix (53Go–55Go). We obtain a time-averaged ß-percentage of 26% (Aß42,Aß40-A21G), 35% (Aß40), and 40% (Aß42-A21G) excluding the first 5-ns and considering residues 1–8 disordered. Circular dichroism and NMR being averaging techniques, it is not possible to determine whether the ß-signal originates from all Aß species or whether some dimeric conformations with high (but not the highest) population exist with a richer ß-composition. In addition, we cannot exclude the possibility that the simulations have not reached equilibrium yet. The second reason is that the effects of the GROMOS force field on the dynamics should be confirmed using other physically based force fields. The two main findings of this study can be summarized as follows.

Firstly, Aß dimers are found in equilibrium between a wide range of topologies, ranging from four-stranded to seven-stranded ß-sheets, with the strands S2 very mobile and the location of the strands S1 fluctuating between residues 11–20 (in Aß40) and residues 13–16 (in Aß40-A21G). This finding raises the question whether an unique inhibitor can block propagation of these structurally distinct dimers into protofibrils.

Secondly, the effect of A21G mutation on Aß dimers is length-dependent and the structures and dynamics of Aß42-A21G cannot be extrapolated from those of Aß40-A21G, and vice versa. This is consistent with earlier experimental studies suggesting that substitutions at positions 22 and 23 produce different effects on Aß assembly depending on whether they occur in Aß40 or Aß42 (56Go). Specifically, we find that the mutation A21G impacts Aß dimers in three ways. A21G destabilizes the ß-sheets and notably strands S2 in Aß40, but not in Aß42. A21G also increases, to a higher extent, the flexibility of the central hydrophobic cluster spanning residues 17–21 in Aß40 than in Aß42, and affects, to various degrees, the populations of the intramolecular and intermolecular salt bridges involving Glu22, Asp23, and Lys28 in Aß40 and Aß42. These three factors likely slow down the formation of higher-order species to direct further assembly into protofibril and could explain the reduced aggregation rate of Aß fibrils containing the Flemish disease-causing mutation.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
We thank R. Tycko for careful reading of the manuscript and for the PDB file of the 2002 Aß40 fibril, and R. Riek for providing us with the coordinates of the Aß42 fibril.

A.H. is supported by the Ministère de l'Education. P.D. thanks the CNRS and Université of Paris 7 for financial support and the Centre Informatique de l'Enseignement Supérieur and Institut du Développement et des Resources en Informatique Scientifique centers for providing computational support.

Submitted on June 8, 2006; accepted for publication July 19, 2006.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
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